Flexible amyloid fiber gel based on albumin and lysozyme as well as preparation method and application of flexible amyloid fiber gel

Abstract

The invention discloses flexible amyloid fiber gel based on albumin and lysozyme as well as a preparation method and application of the flexible amyloid fiber gel, and belongs to the field of delivery of natural high polymer materials and bioactive substances. The method comprises the following steps: respectively preparing ovalbumin fibers (OVAf) and lysozyme fibers (LYZf), mixing according to a volume ratio of 2: 1, adding resveratrol with a final concentration of 100 [mu] mol / L, and incubating at 37 DEG C for 3 h to form flexible gel; according to the gel, ordered crosslinking of fibers is induced through resveratrol, a uniform three-dimensional network is formed, and the gel has good elasticity and flexibility; when being used as a carrier to embed active ingredients such as anthocyanin, the nano-carrier can significantly improve the storage stability and digestion environment tolerance, and has excellent biocompatibility. The invention provides a new scheme for efficient delivery of bioactive components, and can be applied to the fields of functional foods, biological medicines and the like.

Description

Technical Field

[0001] This invention belongs to the field of natural polymer materials and bioactive substance delivery technology, specifically relating to a flexible amyloid fiber gel constructed from albumin (ovalbumin) and lysozyme, its preparation process and its application in the stable delivery of active ingredients. Background Technology

[0002] Amyloid filaments, as an ordered aggregate structure of proteins, have attracted widespread attention in the research of carrier materials in food, medicine, and other fields due to their high mechanical strength, good biocompatibility, and degradability. Ovalbumin (OVA) and lysozyme (LYZ), as naturally derived proteins, have been shown to form amyloid filaments (OVAf, LYZf) that can be used as stabilizers in emulsion preparation. However, single protein filaments have significant drawbacks: Ovalbumin fibers (OVAf) have a linear long-chain structure, which is prone to disordered entanglement and makes it difficult to form a uniform and stable three-dimensional network. Lysozyme filaments (LYZf) are short and rigid, and the system has low viscosity, making it impossible to construct a supportive gel structure on its own. Existing fiber assembly systems lack effective control methods, resulting in low encapsulation rate and poor protection of active ingredients when used as carriers, and they are prone to structural damage in complex environments (such as the gastrointestinal tract).

[0003] The delivery of bioactive ingredients (such as anthocyanins) faces similar challenges: their molecular structures are unstable, easily degraded by temperature, light, and pH, and they are highly water-soluble but poorly lipid-soluble, resulting in low bioavailability. While traditional delivery systems (such as simple protein emulsions and synthetic polymer carriers) can improve their stability to some extent, they suffer from insufficient biocompatibility and weak targeted release capabilities. Therefore, developing a tunable, multifunctional natural protein fiber gel carrier is of great significance for improving the delivery efficiency of bioactive ingredients. Summary of the Invention

[0004] To address the problems of poor controllability of protein amyloid fiber assembly, insufficient gel mechanical properties, low stability and poor bioavailability during the delivery of bioactive components in existing technologies, this invention provides a flexible amyloid fiber gel based on albumin and lysozyme, its preparation method and application.

[0005] The technical solution adopted by this invention to solve its technical problem is: In a first aspect, regarding the preparation method of a flexible amyloid fibrous gel based on albumin and lysozyme, this invention provides a flexible amyloid fibrous gel based on albumin and lysozyme and its preparation method, the specific steps of which are as follows: Step 1, Preparation of ovalbumin amyloid filaments (OVAf): Weigh ovalbumin powder, dissolve it in deionized water to prepare a 3% (w / w) solution, and stir magnetically for 30 min until completely dissolved; adjust the pH of the solution to 2.5 with 1 mol / L hydrochloric acid, and transfer it to a stoppered glass container; place the container in an 85℃ constant temperature water bath for 5 h, stirring once every 30 min to ensure uniform reaction; after heat treatment, cool the solution to room temperature, filter it through a 0.22 μm cellulose filter membrane to remove unreacted protein aggregates, and obtain a clear OVAf solution, which is then refrigerated at 4℃ for later use.

[0006] Step 2, Preparation of lysozyme amyloid cellulose (LYZf): Weigh lysozyme powder, dissolve it in deionized water to prepare a 2% (w / w) solution, and stir magnetically for 20 min until completely dissolved; adjust the pH of the solution to 2.2 with 1 mol / L hydrochloric acid, and transfer it to a stoppered glass container; place the container in an 80℃ constant temperature water bath for 4 h, stirring once every 30 min during the process; after cooling to room temperature, filter through a 0.22 μm cellulose filter membrane to obtain the LYZf solution, and store it at 4℃ for later use.

[0007] Step 3, Preparation of resveratrol-induced OVAf-LYZf composite gel: Take the OVAf solution prepared in Step 1 and the LYZf solution prepared in Step 2 at a volume ratio of 2:1, mix them on a magnetic stirrer for 30 min to obtain a uniform OVAf-LYZf mixture; weigh resveratrol, dissolve it in anhydrous ethanol (final ethanol concentration ≤5%), and slowly add it dropwise to the OVAf-LYZf mixture to make the final concentration of resveratrol in the system 100 μmol / L, and continue stirring for 15 min; transfer the mixture to a petri dish and incubate it in a constant temperature incubator at 37℃ for 3 h to form an elastic transparent gel, which is the flexible amyloid fiber gel based on albumin and lysozyme.

[0008] Secondly, regarding the application of flexible amyloid fibrous gel in the encapsulation of active ingredients, this invention provides an application of flexible amyloid fibrous gel in the encapsulation of active ingredients, the encapsulation and delivery process of which is as follows: Using anthocyanin (cyanidin-3-glucoside) as the model active ingredient, its encapsulation and delivery process is as follows: The flexible amyloid cellulose gel prepared above is used as the dispersed phase and mixed with corn oil at a mass ratio of 9:1. Anthocyanin powder (final concentration of 0.3%) is added and placed in a high-speed shear homogenizer and sheared at 10,000 rpm for 8 minutes to form a uniform O / W type emulsion. This emulsion can stabilize anthocyanin through the spatial barrier effect of the gel network, reducing its degradation during storage and digestion.

[0009] Compared with the prior art, the technical solution of this application has at least the following beneficial effects: The technical solution of this invention achieves ordered assembly and gelation of fibers using resveratrol as a molecular inducer. Through the synergistic effect of hydrophobic interactions and hydrogen bonding, ordered cross-linking of OVAf and LYZf is promoted: OVAf provides a long-chain backbone, and LYZf serves as cross-linking nodes, forming a continuous three-dimensional network structure. Scanning electron microscopy (SEM) observation shows that the composite gel exhibits a uniform porous structure with a pore size distribution of 5-20 μm, which is a significant improvement over the disordered structure of a single fiber system.

[0010] The technical solution of this invention enhances the stability of active ingredients. Using anthocyanins as a model, under storage conditions of 4℃, the anthocyanin retention rate of the composite gel-embedded group was more than 75% after 30 days, which was significantly higher than that of the unembedded group (30%) and the single fiber-embedded group (50%). In the accelerated test at 60℃, the retention rate of the composite gel group still reached 50% after 72 hours, showing excellent thermal stability.

[0011] The technical solution involved in this invention exhibits excellent biocompatibility. The MTT assay was used to detect the toxicity of the gel to Caco-2 cells. The results showed that the cell survival rate was higher than 90% in the concentration range of 0.1-10 mg / mL, which meets the safety requirements of biomaterials. Attached Figure Description

[0012] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the embodiments and comparative examples will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0013] Appendix Figure 1 Particle size distribution (A), zeta potential (B), atomic force microscopy, and appearance characteristics (C) of resveratrol-induced ovalbumin and lysozyme nanofiber gels. OVAf represents ovalbumin fibers, LYZf represents lysozyme fibers, Mix represents a mixture of ovalbumin and lysozyme fibers, and 50 to 200 represents resveratrol concentration in μM.

[0014] Appendix Figure 2 THT fluorescence spectrum (A), intrinsic fluorescence spectrum (B), XRD (C), (D) resveratrol-induced ovalbumin and lysozyme nanofiber gel. OVAf represents ovalbumin fiber, LYZf represents lysozyme fiber, Mix represents a mixture of ovalbumin and lysozyme fibers, and 50 to 200 represents resveratrol concentration in μM.

[0015] Appendix Figure 3FTIR spectra (A), intermolecular interactions (B), secondary structure (C), and hydrogen bonds (D) of glucosidin-induced ovalbumin and lysozyme nanofiber gels. OVAf represents ovalbumin fibers, LYZf represents lysozyme fibers, Mix represents a mixture of ovalbumin and lysozyme fibers, and 50 to 200 represents glucosidin concentration in μM.

[0016] Appendix Figure 4 The surface hydrophobicity (A), droplet imaging (B), surface pressure (C), and diffusion rate (D) of resveratrol-induced ovalbumin and lysozyme nanofiber gels are shown. OVAf represents ovalbumin fibers, LYZf represents lysozyme fibers, Mix represents a mixture of ovalbumin and lysozyme fibers, and 50 to 200 represent resveratrol concentrations in μM.

[0017] Appendix Figure 5 Appearance characteristics of the emulsion during storage (A), optical and CLSM images of emulsion droplets (B), particle size distribution (C) and average particle size (D, D3, 2, D4, 3) of the anthocyanin-loaded emulsion stabilized by nanofiber gel. OVAf represents ovalbumin fiber, LYZf represents lysozyme fiber, Mix represents a mixture of ovalbumin fiber and lysozyme fiber, and 50 to 200 represent resveratrol concentration in μM.

[0018] Appendix Figure 6 Specific surface area (A), average particle size (B), apparent viscosity (C), and frequency scan (D) of anthocyanin-loaded nanofiber gel stabilized emulsions. OVAf represents ovalbumin fiber, LYZf represents lysozyme fiber, Mix represents a mixture of ovalbumin fiber and lysozyme fiber, and 50 to 200 represent resveratrol concentration in μM.

[0019] Appendix Figure 7 The values ​​represent the average particle size (A), zeta potential (B), CLSM microstructure (C), and bioaccessibility (D) of oxalate during in vitro digestion in nanofiber gel-stabilized emulsions, and the micelle uptake (E) of anthocyanins after 120 minutes of culture in a Caco-2 cell monolayer membrane. OVAf represents ovalbumin filaments, LYZf represents lysozyme filaments, Mix represents a mixture of ovalbumin filaments and lysozyme filaments, and 50 to 200 represent resveratrol concentrations in μM.

[0020] Appendix Figure 8Cell viability (A), mean intensity of reactive oxygen species (ROS) fluorescence (B), staining of live and dead cells using AM and PI (C), and ROS fluorescence imaging (D), SOD (E) and MDA (F) content of Caco-2 cells after stimulation with 100 mM hydrogen peroxide for 24 hours, along with digested micelles containing emulsion. OVAf represents ovalbumin filaments, LYZf represents lysozyme filaments, Mix represents a mixture of ovalbumin filaments and lysozyme filaments, and 50 to 200 represents resveratrol concentration in μM. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0022] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to specific embodiments.

[0023] In a first aspect, regarding the preparation method of a flexible amyloid fibrous gel based on albumin and lysozyme, this invention provides a flexible amyloid fibrous gel based on albumin and lysozyme and its preparation method, the specific steps of which are as follows: Step 1, Preparation of ovalbumin amyloid filaments (OVAf): Weigh ovalbumin powder, dissolve it in deionized water to prepare a 3% (w / w) solution, and stir magnetically for 30 min until completely dissolved; adjust the pH of the solution to 2.5 with 1 mol / L hydrochloric acid, and transfer it to a stoppered glass container; place the container in an 85℃ constant temperature water bath for 5 h, stirring once every 30 min to ensure uniform reaction; after heat treatment, cool the solution to room temperature, filter it through a 0.22 μm cellulose filter membrane to remove unreacted protein aggregates, and obtain a clear OVAf solution, which is then refrigerated at 4℃ for later use.

[0024] Step 2, Preparation of lysozyme amyloid cellulose (LYZf): Weigh lysozyme powder, dissolve it in deionized water to prepare a 2% (w / w) solution, and stir magnetically for 20 min until completely dissolved; adjust the pH of the solution to 2.2 with 1 mol / L hydrochloric acid, and transfer it to a stoppered glass container; place the container in an 80℃ constant temperature water bath for 4 h, stirring once every 30 min during the process; after cooling to room temperature, filter through a 0.22 μm cellulose filter membrane to obtain the LYZf solution, and store it at 4℃ for later use.

[0025] Step 3, Preparation of resveratrol-induced OVAf-LYZf composite gel: Take the OVAf solution prepared in Step 1 and the LYZf solution prepared in Step 2 at a volume ratio of 2:1, mix them on a magnetic stirrer for 30 min to obtain a uniform OVAf-LYZf mixture; weigh resveratrol, dissolve it in anhydrous ethanol (final ethanol concentration ≤5%), and slowly add it dropwise to the OVAf-LYZf mixture to make the final concentration of resveratrol in the system 100 μmol / L, and continue stirring for 15 min; transfer the mixture to a petri dish and incubate it in a constant temperature incubator at 37℃ for 3 h to form an elastic transparent gel, which is the flexible amyloid fiber gel based on albumin and lysozyme.

[0026] Secondly, regarding the application of flexible amyloid fibrous gel in the encapsulation of active ingredients, this invention provides an application of flexible amyloid fibrous gel in the encapsulation of active ingredients, the encapsulation and delivery process of which is as follows: Using anthocyanin (cyanidin-3-glucoside) as the model active ingredient, its encapsulation and delivery process is as follows: The flexible amyloid cellulose gel prepared above is used as the dispersed phase and mixed with corn oil at a mass ratio of 9:1. Anthocyanin powder (final concentration of 0.3%) is added and placed in a high-speed shear homogenizer and sheared at 10,000 rpm for 8 minutes to form a uniform O / W type emulsion. This emulsion can stabilize anthocyanin through the spatial barrier effect of the gel network, reducing its degradation during storage and digestion.

[0027] Example 1: Preparation and characterization of flexible amyloid fibrous gel OVAf preparation: Weigh 3g of ovalbumin, dissolve it in 97mL of deionized water, stir until dissolved, adjust the pH to 2.5, heat in an 85℃ water bath for 5h, cool and filter, and store at 4℃.

[0028] Preparation of LYZf: Weigh 2g of lysozyme, dissolve it in 98mL of deionized water, stir until dissolved, adjust the pH to 2.2, heat in an 80℃ water bath for 4h, cool and filter, and store at 4℃.

[0029] Preparation of composite gel: Mix 20 mL of LOVAf with 10 mL of LYZf, add resveratrol (final concentration 100 μmol / L), and incubate at 37 °C for 3 h to form a gel.

[0030] Characterization results: Fourier transform infrared spectroscopy (FTIR) showed that the gel exhibited enhanced absorption at 1620 cm⁻¹ (the characteristic peak of β-sheet), indicating that the amyloid fiber structure was stable. Atomic force microscopy (AFM) revealed that the fibers had a diameter of 8-12 nm and a length of 1-5 μm, forming an interwoven network.

[0031] Example 2: Preparation and stability testing of anthocyanin emulsions based on composite gels Emulsion preparation: Take 18g of composite gel, 2g of corn oil and 0.06g of anthocyanins, mix them and shear at 10000rpm for 8min to obtain the emulsion.

[0032] Stability test: Centrifugation stability: No stratification was observed after centrifugation at 3000 rpm for 15 minutes; pH stability: Particle size change rate of emulsion <10% within pH range of 3-9; Digestive stability: After treatment in a simulated gastric juice environment (pH 1.2, 37℃) for 2 hours, the emulsion interface remained intact, and the anthocyanin release rate was <15%.

[0033] The embodiments described above are some, but not all, embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments of the invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. A method for preparing a flexible amyloid fibrous gel based on albumin and lysozyme, characterized in that, Includes the following steps: Step 1. Preparation of ovalbumin fiber (OVAf): Dissolve ovalbumin in deionized water to prepare a 3% (w / w) solution, adjust the pH to 2.5, heat treat at 85℃ for 5 hours, and filter to obtain OVAf; Step 2. Preparation of lysozyme fiber (LYZf): Dissolve lysozyme in deionized water to prepare a 2% (w / w) solution, adjust the pH to 2.2, heat treat at 80℃ for 4 hours, and filter to obtain LYZf; Step 3. Preparation of composite gel: OVAf and LYZf are mixed at a volume ratio of 2:1, and resveratrol is added to make the final concentration 100 μmol / L. The mixture is incubated at 37℃ for 3 h to obtain flexible amyloid fiber gel.

2. The method for preparing a flexible amyloid fiber gel based on albumin and lysozyme according to claim 1, characterized in that, In steps 1 and 2, the mixture was stirred once every 30 minutes during the heat treatment, and a 0.22 μm cellulose filter membrane was used for filtration.

3. The method according to claim 1, characterized in that, In step 3, resveratrol is dissolved in anhydrous ethanol and then added dropwise, with the final concentration of ethanol in the system ≤5%.

4. A flexible amyloid fiber gel based on albumin and lysozyme prepared by the method according to any one of claims 1-3.

5. The application of the gel according to claim 4 in the encapsulation of active ingredients, characterized in that, The gel was mixed with vegetable oil at a mass ratio of 9:1, and anthocyanins were added to a final concentration of 0.3%. The mixture was sheared at 10,000 rpm for 8 minutes to obtain an anthocyanin-loaded O / W emulsion.

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